Xenorhabdus sp. and Photorhabdus sp. strains have previously been shown to produce an array of extracellular proteins and small molecules or secondary metabolites having specialized functions. Among the more commercially interesting are proteins and small molecules having antibiotic properties or proteins which exhibit insect inhibitory activity. A small number of insect inhibitory proteins have previously been identified from these bacteria, symbionts of insect-parasitic nematodes. In view of the biotechnology methods which are now available, such proteins and compositions have great potential for use as biologically safe and effective pest control agents. Unlike chemical pesticide compositions, these proteins have no effect upon the environment in general, can be targeted to direct their effect primarily upon target insect species, and have no effect on non-target species. These proteins are comparable in nature to Bacillus thuringiensis (BT) proteins, which are the most widely used biological insect pest control agents derived from various strains of Bacillus thuringiensis. BT compositions have been in commercial use for more than twenty years as topically applied insect control agents and more recently genes encoding various BT proteins have been expressed in transgenic plants, and in particular in agronomically important crops such as soybean, corn, wheat, rice, and cotton. However, one issue related to the use of BT proteins is resistance management. The concern is that target insect pests feeding on a plant expressing a single BT protein that is generally effective against that pest species will develop resistance to the protein in some calculable period of time. The answer to this problem has been to include in the plant another BT protein also toxic to the same target pest species. The idea is similar in nature to bacterial resistance management, in that the development of resistance to either of the BT proteins will be delayed because pest will not produce progeny that are resistant to either of the BT proteins, in particular if the two proteins that are expressed in the plant have different modes of action or bind different receptors in the insect midgut. Unfortunately, BT proteins are highly related and often it is difficult to distinguish whether two BT proteins toxic to the same insect species have different modes of action. Thus, even though a great variety of BT proteins have been identified, characterized and categorized into distinct classes of proteins, all appear to act in a very similar fashion. Therefore, a different resistance management strategy which takes advantage of insect inhibitory proteins derived from distinct microbial sources other than Bacillus thuringiensis would be desirable. Insect inhibitory proteins isolated from Xenorhabdus and Photorhabdus species of bacteria seem to have all the prerequisites for the delivery of novel genes for transgenic expression of insect pest inhibiting proteins to provide pest resistance to plants, either alone or in combination with Bacillus thuringiensis insecticidal crystal proteins.
Xenorhabdus sp. is a Gram-negative bacterium, member of the family of Enterobacteriaceae, and symbiotically associated with nematodes of the genus Steinernema. The nematode-bacterial complex can be characterized as an obligate and lethal parasitic relationship, specializing in parasitizing and proliferating in soil insect larvae. Infective, non-feeding stages of these nematodes live in soil and carry their nematode-genus-specific symbiotic bacteria in the gut. It is believed that the nematodes actively search for the appropriate insect host, invade the insect larvae through natural openings or lesions in the cuticle and, once inside the hemolymphe, release their symbiotic bacteria. The nematode-bacterial complex secretes a variety of highly efficient extracellular metabolites and proteins exhibiting insectic inhibitory, bactericidal, fungicidal and nematicidal properties to secure the larval mass as a source of nutrition. An array of extracellular enzymes such as lipases, phospholipases, proteases, nucleases as well as several broad spectrum antibiotics, and antifungal and nematicidal compositions are also secreted (Boemare & Akhurst, J. Gen. Microbiol. 134: 751-761 (1988); Li et al., Can. J. Microbiol. 43(8):770-773 (1997); McInerney et al., J. Nat. Prod. 54(3):774-84 (1991); McInerney et al., J. Nat. Prod. 54(3):785-95 (1991); Sundar and Chang, J. Gen. Microbiol. 139 (Pt 12):3139-48 (1993)). It has been discovered that some compounds secreted by Xenorhabdus exhibit anti-neoplastic (U.S. Pat. No. 5,827,872), acaricidal, anti-inflammatory and anti-ulcerogenic properties (U.S. Pat. No. 4,837,222). U.S. Pat. No. 6,048,838 describes insect inhibitory proteins which exhibit a molecular weight of greater than 100 kDa produced by Xenorhabdus sp. which are orally active against a variety of insect species including the orders Lepidoptera, Coleoptera, Diptera, and Acarina.
The nomenclature and taxonomic characterization of Xenorhabdus has recently been subject to innovations in the state of the art. The genus Photorhabdus was separated from the genus Xenorhabdus in 1993 because of significant differences in biochemical and molecular characterization (Boemare et al., Int. J. Syst. Bacteriol. 43: 249-255 (1993)). Xenorhabdus exists of at least 4 known species: X. nematophilus, X. beddingii, X. poinarii and X. bovienii. (Brunel et al., Appl. & Environm. Microbiol. 63: 574-580 (1997)). Species of Xenorhabdus as well as Photorhabdus species can be distinguished from each other by restriction analysis of thermally amplified 16S rRNA genes (Brunel et al., Appl. Environm. Microbiol. 63: 574-580 (1997)).
The genetic diversity of symbiotic Xenorhabdus and Photorhabdus bacteria associated with entomopathogenic nematodes appears to be quite large. The genus Xenorhabdus appears more diverse than the genus Photorhabdus, and for both genera, the bacterial genotype diversity is in congruence with the host-nematode taxonomy. It has been found that the occurrence of symbiotic bacterial genotypes was related to the ecological distribution of host nematodes (Fisher-Le Saux et al., Appl. Environ. Microbiol. 64(11):4246-54 (1998)). Xenorhabdus bacteria isolated from the same geographical location seem to be more similar to each other, regardless of nematode species than bacteria from one nematode species found in very diverse geographical locations (Liu et al., Intl. J. Syst. Bacteriol. 47:948-951; 1997).
Therefore, there is a great deal of interest in identifying the genes that encode new insect inhibiting proteins, and proteins involved in the biosynthetic pathways of novel antibiotics produced by Xenorhabdus and Photorhabdus bacteria, as well as other useful proteins. Sequencing of the entire genome of Xenorhabdus would facilitate such an endeavor, because it would allow dissection and analysis of the genome into discrete genes encoding proteins having beneficial properties as described herein.